Uses of antileukoprotease in carcinoma

ABSTRACT

The present invention provides a method of detecting/monitoring tumor growth and progression in a tissue by measuring the level of antileukoprotease. Also provided is a method of treating an individual having a tumor by administering antileukoprotease to inhibit the activity of stratum corneum chymotryptic enzyme. Specifically, the tumor is an ovarian carcinoma.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This non-provisional patent application claims benefit ofprovisional patent application U.S. Serial No. 60/159,972, filed Oct.18, 1999, now abandoned.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the fields of proteinchemistry and cancer therapy. More specifically, the present inventionrelates to antileukoprotease, a peptide inhibitor of stratum corneumchymotryptic enzyme, and its uses in carcinoma diagnosis and treatment.

[0004] 2. Description of the Related Art

[0005] Proteases mediate specific proteolysis involved in processing ofprecursors of protein hormones, activation of regulatory enzymes inblood coagulation and complement activation, and the tissuerearrangement involved in tumor progression (1). In the process of tumorinvasion and metastasis, proteases mediate the digestion of neighboringextracellular matrix components during initial tumor growth. This allowsshedding of minor cells into the surrounding environment, providing thebasis for invasion of basement membranes in target metastatic organs.Proteolytic digestion is also required for release and activation ofmany growth and angiogenic factors (2-4).

[0006] A large number of reports have demonstrated increased productionof several classes of proteases, including matrix metalloproteases(MMP's), cysteine proteases, aspartic pro teases and serine proteases intumor cells (5-9). The proteolysis of the extracellular matrix is ahighly complicated process, which probably involves a cascade of eventsrequiring a variety of proteases (10). In this cascade, the integratedcapacity for extracellular matrix digestion, tumor cell invasion, andmetastatic growth may be mediated by proteases with uniquespecificities. This hypothesis is supported by findings that some agentsspecifically inhibit one of these proteases to reduce tumor cellinvasion (11,12).

[0007] Stratum corneum chymotryptic enzyme (SCCE) was originallyisolated from a keratinocyte derived library and was identified as aserine protease (13,14). Analysis of mRNA showed that two transcripts of1.2 kb and 2.0 kb were present, and abundant expression of the stratumcorneum chymotryptic enzyme gene was restricted to human skin.Immunohistochemical studies confirmed that stratum corneum chymotrypticenzyme was a tissue-specific enzyme only expressed by the stratumcorneum (15). The nucleotide sequence includes an open reading frame fora stratum corneum chymotryptic enzyme precursor protein consisting of253 amino acids. This inactive precursor becomes proteolytically activeafter tryptic removal of a 7 amino acid peptide from the amino terminalend of the propeptide. Recent studies have revealed that stratum corneumchymotryptic enzyme appears to catalyze the degradation of intercellularcohesive structures between corneocytes in the outermost cornified layerof the skin and contributes to the cell shedding process at the skinsurface (14, 16, 17). This process occurs possibly through thedegradation of matrix components including the desmosomal proteindesmoglein I.

[0008] Protease inhibitor antileukoprotease (ALP), also known assecretory leukocyte proteinase inhibitor (SLPI), has been identified asa potent inhibitor of leukocyte elastase, cathepsin G, chymotrypsin andtrypsin (18). Antileukoprotease has been cloned from skin tissue andshown to be a specific inhibitor of the stratum corneum chymotrypticenzyme (SCCE) (17). This serine protease is produced and released intomucus by secretory cells in the parotid, bronchus, cervix and testicularglands (18). There, it is thought to play a physiological role inpreventing the proteolytic degradation of these tissues. However, littlehas been known about the expression of antileukoprotease in human cancertissues, including ovarian cancer.

[0009] The prior art is deficient in the lack of effective means ofusing antileukoprotease as a diagnostic or monitoring tool ofcarcinomas. The present invention fulfills this long-standing need anddesire in the art.

SUMMARY OF THE INVENTION

[0010] The present invention demonstrates that antileukoprotease (ALP)is overexpressed in low malignant potential tumors and carcinomas inovary, while little or no transcript is present in normal adult andfetal tissues. This indicates that antileukoprotease may be used as adiagnostic or monitoring tool of ovarian tumors.

[0011] In one embodiment of the present invention, there is provided amethod of detecting growth of an ovarian or ovarian-derived metastatictumor in an individual suspected to have such a tumor, comprising thestep of detecting the level of antileukoprotease in a test tissue, asecretion from a test tissue or the blood. If the level exceeds the meanbasal level of antileukoprotease in nondiseased individuals by 2standard deviation or more, the individual has ovarian and ovarianderived metastatic tumor growth.

[0012] In still another embodiment of the present invention, there isprovided a method of treating an individual having a ovarian tumor byadministering antileukoprotease to the individual.

[0013] In yet another embodiment of the instant invention, a method ofpreventing metastasis of an ovarian tumor is provided whereinantileukoprotease is administered to an individual having such a tumor.

[0014] Other and further aspects, features, and advantages of thepresent invention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] So that the matter in which the above-recited features,advantages and objects of the invention, as well as others which willbecome clear, are attained and can be understood in detail, moreparticular descriptions of the invention briefly summarized above may behad by reference to certain embodiments thereof which are illustrated inthe appended drawings. These drawings form a part of the specification.It is to be noted, however, that the appended drawings illustratepreferred embodiments of the invention and therefore are not to beconsidered limiting in their scope.

[0016] FIGS. 1A-1D show the results of northern blot analysis ofantileukoprotease expression in various tissues. FIG. 1A confirms thepresence of antileukoprotease transcript in tumor tissues as opposed tonormal tissues. ALP mRNA was detected as a 1.4 kb transcript in ovariancancers but was not detected in normal ovary. FIG. 1B shows that theantileukoprotease transcript was not detected in fetal tissues. FIG. 1Cand FIG. 1D show that antileukoprotease transcript was not detected inmany normal adult tissues but was abundantly expressed in othersincluding lung.

[0017]FIG. 2 shows the overexpression of antileukoprotease in ovariancarcinoma specimens as detected by quantitative PCR of antileukoproteasetranscript. Expression levels of antileukoprotease relative to β-tubulinare significantly elevated in many cancer samples relative to levelsobserved in normal ovary.

[0018]FIG. 3 shows a graphical representation ofantileukoprotease/β-tubulin ratios in normal ovaries, LMP tumors, andcarcinomas. ALP mRNA expression levels were significantly elevated inLMP tumors and carcinomas as compared with levels in normal ovaries.

[0019] FIGS. 4A-4E show immunohistochemical staining results forantileukoprotease in various ovarian tumors. FIG. 4A showsimmunohistochemical staining or normal uterine endocervix at 50×magnification. The endocervix section acts as a positive control asnormal endocervical glandular cells and the mucin in the endocervicalglands is positive for ALP. FIG. 4B, showing a 100× magnification ofnormal ovarian surface epithelium, is negative for antileukoproteaseimmunohistochemical staining. Positive antileukoproteaseimmunohistochemical staining was observed in both the cell membrane andcytoplasm of ovarian cancer cells of FIG. 4C (endometrioidadenocarcinoma, X50). In FIG. 4D (mucinous adenocarcinoma, X50), themucin as well as tumor glands of a mucinous carcinoma were positive forantileukoprotease staining. Positive staining for antileukoprotease wasalso observed in secretions from the clear cell carcinoma shown in FIG.4E (clear cell carcinoma, X50) and in the serous carcinomas shown inFIGS. 4F and 4G.

DETAILED DESCRIPTION OF THE INVENTION

[0020] This invention encompasses a demonstration of overexpression ofantileukoprotease (ALP) in carcinoma tissues. It shows that many lowmalignant potential tumors and most carcinomas tested have astatistically significant overexpression of antileukoprotease.Antileukoprotease is a secreted protein with a distribution of cysteinesknown to provide extraordinary stability to protease inhibitors. Thismolecule therefore fulfills many criteria necessary for a valuablemarker of tumor growth and progression. Antileukoprotease isoverexpressed in tumor cells secreted to the extracellular space and isa relatively small protein (approximately 100 amino acids) providinggood opportunity for uptake into the circulation. Moreover,antileukoprotease is a very stable protein thus providing the potentialfor a relatively long half life in the circulation.

[0021] In one embodiment of the present invention, there is provided amethod of detecting growth of an ovarian or ovarian derived metastatictumor in an individual suspected of having such a tumor, comprising thestep of detecting the level of antileukoprotease in a test sample,wherein if the level exceeds the mean basal level of antileukoproteasein nondiseased individuals by 2 standard deviation and greater, theindividual has growth in an ovarian or ovarian-derived tumor.Preferably, the tumor may be a low malignant potential tumor or acarcinoma. Examples of carcinoma include serous carcinoma, mucinouscarcinoma, endometrioid carcinoma and clear cell carcinoma. The testsamples may be an ovarian secretion, an ovarian biopsy, a metastatictumor biopsy and blood. The antileukoprotease may be detected by variousmeans know to one having ordinary skill in this art including westernblot analysis, immunohistochemical staining, or other immunochemicalmethods. In still another embodiment of the present invention, there isprovided a method for treating an individual having an ovarian orovarian-derived metastatic tumor, comprising the step of administeringantileukoprotease to the individual. Preferably, the tumor may be a lowmalignant potential tumor or an ovarian carcinoma. If it is an ovariancarcinoma, the tumor may be a serous carcinoma, a mucinous carcinoma, anendometrioid carcinoma and a clear cell carcinomas. Theantileukoprotease may be administered systemically or locally.

[0022] In yet another embodiment of the present invention, there isprovided a method for method of preventing metastasis of an ovariantumor or ovarian derived metastatic tumor comprising the step ofadministering antileukoprotease to an individual having such a tumor.Preferably, the tumor may be a low malignant potential tumor or anovarian carcinoma. If it is an ovarian carcinoma, the tumor may be aserous carcinoma, a mucinous carcinoma, an endometrioid carcinoma and aclear cell carcinomas. The antileukoprotease may be administeredsystemically or locally.

[0023] The following examples are given for the purpose of illustratingvarious embodiments of the invention and are not meant to limit thepresent invention in any fashion.

EXAMPLE 1

[0024] Tissue Samples

[0025] Fresh surgical specimens of ovarian tumors were obtained from lowmalignant potential tumors and carcinomas. Clinical staging wasdetermined according to the criteria of the International Federation ofGynecology and Obstetrics (FIGO). Normal ovaries were obtained frompatients who underwent surgery for benign gynecological disease. Thematerials were obtained immediately after the surgical procedures andfrozen in liquid nitrogen and stored at −80° C. prior to mRNA isolation.Two ovarian cancer cell lines, SW626 and CaOv3, were also used.

EXAMPLE 2

[0026] mRNA Extraction and cDNA Synthesis

[0027] Extraction of mRNA from tissue specimens and cDNA synthesistherefrom were carried out by the methods described previously (19).mRNA was isolated by using a RiboSep™ mRNA isolation kit (BectonDickenson Labware). In this procedure, poly A+ mRNA was isolateddirectly from the tissue lysate using the affinity chromatography mediaoligo(dT) cellulose. The amount of mRNA recovered was quantitated by UVspectrophotometry. The cDNA was synthesized with either 2.0 μg or 5.0 μgof mRNA by random hexamer priming using a 1st strand™ cDNA synthesis kit(CLONTECH). The efficiency of the cDNA synthesis was estimated usingglucose 3-phosphate dehydrogenase (G3PDH) amplimers (Clontech, PaloAlto, Calif., USA) as a positive control.

EXAMPLE 3

[0028] Quantitative PCR

[0029] mRNA overexpression of antileukoprotease was determined using aquantitative PCR approach as previously reported (19-21). The ALP targetsequences were amplified in parallel with the β-tubulin gene. β-tubulinhas previously been established to be a consistently expressed internalcontrol for both normal and tumor tissues (19,21). The followingspecific oligonucleotide primers were used for antileukoproteaseamplification by PCR: Primer set 1- forward 5′-TGCATTGACAACGAGGC-3′ (SEQID No. 1) and reverse 5′-CTGTCTTGACATTGTTG -3′ (SEQ ID No. 2); and,primer set 2-forward 5′-CCTTCAAAGCTGGAGTCTGT-3′ (SEQ ID No. 3) andreverse (CCAAAGGAGGATATCAGTGG-3′ (SEQ ID No. 4). The primers for theβ-tubulin internal control amplification were, forward5′-CGCATCAACGTGTACTACAA-3′ (SEQ ID No. 5) and reverse5′-TACGAGCTGGTGGACTGAGA-3′ (SEQ ID No. 6). The predicted sizes of theamplified gene were 400 bp for ALP primer set 1, 469 bp for ALP primerset 2 and 454 bp for β-tubulin. The primer sequences used in this studywere designed according to the cDNA sequences described forantileukoprotease and by Hall et al. for β-tubulin (22).

[0030] The PCR reaction mixture consisted of cDNA derived from 50 ng ofmRNA, 5 pmol of sense and antisense primers for both the stratum corneumchymotryptic enzyme gene and the β-tubulin gene, 200 μmol of dNTPs, 5μCi of [α-³²P]dCTP and 0.25-0.625 units of Taq DNA polymerase withreaction buffer (Promega) in a final volume of 25 μl. The targetsequences were amplified in parallel with the β-tubulin gene. Thirtycycles of PCR were carried out in a Thermal Cycler (Perkin-Elmer Cetus,Foster City, Calif., USA). Each cycle of PCR included 30 seconds ofdenaturation at 95° C., 30 seconds of primer annealing at 58-60° C. and30 seconds of extension at 72° C. It was previously established (19,21)and confirmed for the stratum corneum chymotryptic enzyme thatco-amplification with β-tubulin under these conditions for 30 cyclesremains linear for both products. Tubes containing all ingredientsexcept templates were included in all runs as negative controlreactions.

[0031] The PCR products were separated on 2% agarose gels and theradioactivity of each PCR product was determined by using a PhosphoImager (Molecular Dynamics). In the present study, the gene expressionof the stratum corneum chymotryptic enzyme was calculated as the ratioof ALP to β-tubulin as measured by the phospho imager. Theoverexpression cut-off value was defined at two standard deviation abovethe mean expression level for normal ovarian expression.. Forstatistical analysis, the χ² test and Fischer's exact probability wereused for contingency analysis and the unpaired student's T-test was usedfor the comparison of the mean values of normal ovary and tumors.Significance was defined as p<0.05.

EXAMPLE 4

[0032] Northern Blot Analysis

[0033] Ten μg of mRNA were loaded onto a 1% formaldehyde-agarose gel,resolved by electrophoresis and blotted on a Hybond-N+ nylon membrane(Amersham, Amersham, UK). ³²P-labeled cDNA probes were made byPrime-a-Gene Labeling System (Promega, Madison, Wis.). The PCR productswere amplified using the specific primers described above as probes. Theblots were prehybridized for 30 min and hybridized for 60 min at 68° C.with a ³²P-labeled cDNA probe in ExpressHyb Hybridization Solution(CLONTECH, Palo Alto, Calif.). Control hybridization to determinerelative gel loading was performed with the β-tubulin probe.

[0034] Normal human tissues including spleen, thymus, prostate, testis,ovary, small intestine, colon, peripheral blood leukocyte, heart, brain,placenta, lung, liver, skeletal muscle, kidney, pancreas and normalhuman fetal tissues; brain, lung, liver and kidney (Human MultipleTissue Northern Blot; CLONTECH, Palo Alto, Calif.) were all examinedusing the same hybridization procedure.

EXAMPLE 5

[0035] Immunohistochemistry

[0036] Polyclonal rabbit antibodies were generated by immunization witha poly-lysine linked multiple antigen peptide (a 12 amino acid sequencenear the carboxy terminal end of ALP). Immunohistochemical localizationof antileukoprotease antigen was examined using normal ovaries, mucinousLMP tumor and adenocarcinomas (including serous adenocarcinomas,mucinous adenocarcinoma and clear cell carcinomas) in the same series ofthe samples as were used for mRNA isolation. Formalin fixed andparaffin-embedded sections, 4 μm thick, were cut and mounted onaminopropyltriethoxysilane treated slides. Slides were routinelydeparaffinized with xylene and rehydrated with a series of ethanolwashes. Nonenzymatic antigen retrieval was performed by microwave heattreatment seven times for three minutes in a 0.01 M sodium citratebuffer (pH 6.0). Immunohistochemical staining was performed manuallyusing the avidin-biotin peroxidase complex technique (Vectastain EliteABC kit, Vector Laboratories, Burlingame, Calif., USA).

[0037] This indirect immunoperoxidase staining procedure was performedat room temperature. Endogenous peroxidase and nonspecific backgroundstaining were blocked by incubating the slides with methanol containing0.3% H₂O₂ for 30 minutes. The slides were washed with phosphate-bufferedsaline (PBS) for 30 minutes, blocked with normal goat serum for thirtyminutes, and incubated with the above rabbit anti-antileukoproteasepolyclonal antibody for two hours. After washing with phosphate-bufferedsaline (PBS) for 30 minutes, sections were incubated with biotinylatedanti-rabbit IgG for 30 minutes. After washing with PBS for 30 minutes,slides were incubated with ABC reagent for 30 minutes. The finalproducts were visualized using the 3-amino-9-ethylcarbazole (AEC)substrate system (DAKO Corporation, Carpinteria, Calif.) and werecounterstained with Mayer hematoxylin for 20 seconds before mounting.Positive controls and negative controls were used for each section.Normal endocervix was used as a positive control. Negative controls wereprepared by using normal rabbit serum on sections instead of the primaryantibody. All experiments were duplicated. The stained slides wereexamined microscopically by 3 observers. The presence of more than 10%focally distributed positive tumor cells was the criterion for a 1+positive staining and more than 50% of positive tumor cells was thecriterion for a 2+ positive staining. When less than 10% of the cellsshowed positive nuclear staining, the staining was considered negative

EXAMPLE 6

[0038] Western Blot

[0039] Approximately 20 ng of MDA-MBA-435S and HeLa cell lysates wereseparated on a 15% SDS-PAGE gel and electroblotted to PVDF at 100 V for40 minutes at 4° C. The proteins were fixed to the membrane byincubation in 50% MeOH for 10 minutes. The membrane was blockedovernight in TBS, pH 7.8 containing 0.2% non-fat milk. Primary antibodywas added to the membrane at a dilution of 1:100 in 0.2% milk/TBS andincubated for 2 hours at room temperature. The blot was washed andincubated with a 1:3000 dilution of alkaline-phosphatase conjugated goatanti-rabbit IgG (BioRad) for one hour at room temperature. The blot waswashed and incubated with a chemiluminescent substrate before a 10second exposure to X-ray film for visualization.

EXAMPLE 7

[0040] Northern blot analysis of antileukoprotease expression in ovariancarcinomas

[0041] To evaluate the mRNA expression of antileukoprotease in ovariantumors and to examine the size of the mRNA transcript, Northern blotanalysis with a ³²P-labeled antileukoprotease probe was performed inrepresentative cases of each type of ovarian carcinoma (FIG. 1A).Northern blot analysis revealed a 1.4 kb transcript in all of thesubtypes of ovarian carcinoma including serous, mucinous, endometrioid,and clear cell carcinomas (FIG. 1A, lanes 2-4). No transcript wasobserved in normal ovarian tissue (FIG. 1A, lane 1). These resultsdemonstrate that an appropriately sized transcript of theantileukoprotease gene is expressed in the ovarian carcinomas.

EXAMPLE 8

[0042] Northern blot analysis of antileukoprotease expression in normaltissues

[0043] Northern blot analysis with a ³²P-labeled antileukoprotease probewas also performed in normal fetal and adult tissue samples. In thefetal tissues, little or no antileukoprotease expression was detected infetal brain, lung, liver, kidney and pancrease (FIG. 1B).Antileukoprotease transcript was also not detected in many normal adulttissues, including heart, brain, placenta, liver, skeletal muscle,kidney, and pancreas (FIGS. 1C and 1D). Only adult lung showed anabundance of the antileukoprotease transcript (FIG. 1D). Lower levels ofexpression were observed in adult prostate, ovaries, and the colon (FIG.1C).

[0044] Further data supporting tumor expression of antileukoprotease wasobtained using polyclonal antibodies developed to multiple antigenpeptide (MAP peptide), a 12 amino acid sequence near the carboxylterminal of antileukoprotease (data not shown).

EXAMPLE 9

[0045] Semi-quantitative PCR analysis of Antileukoprotease (ALP)Overexpression.

[0046] To confirm the results of the northern blot analysis,semi-quantitative PCR was performed with primers for antileukoproteaseand the β-tubulin internal control on 34 ovarian carcinomas and 10normal ovarian tissue samples. FIG. 2 shows an example of asemi-quantitative PCR evaluation of antileukoprotease expression usingoligonucleotides specific for antileukoprotease and the β-tubulininternal control. It was apparent that many ovarian carcinoma samples,when compared to normal ovarian samples, exhibit elevated levels ofantileukoprotease transcript relative to the levels of the controlβ-tubulin transcript, providing additional evidence thatantileukoprotease is often overexpressed in ovarian carcinoma specimens.

[0047] The relative expression antileukoprotease of ALP and β-tubulin ineach sample were measured by phospho imager analysis. The results foreach tissue sample are presented in Table 1. It can be noted that manylow malignant potential tumors and most carcinomas have a statisticallysignificant overexpression of antileukoprotease. In many cases, thisexpression exceeds the mean for normal by 4SD. It should also be notedthat in at least one ovarian tumor cell line, CAOV3, significantoverexpression of antileukoprotease was observed (Table 1). TABLE 1 Acomplete analysis of expression comparing normal ovarian tissue to lowmalignant potential tumors and to overt carcinomas Lab No. Hist. TypeALP alp 456 normal ovary 0.0033   0 856 normal ovary 0.0228   0 858normal ovary 0.0246   0 1235 normal ovary 0.04   0 1338 normal ovary0.0997   0 1339 normal ovary 0.1058   0 1343 normal ovary 0.0433   01344 normal ovary 0.1014   0 1345 normal ovary 0.069   0 2296 normalovary 0.0146   0 481 s adenoma (LMP) 0.3214   4+  1448 s adenoma (LMP)0.0251   0 1452 s adenoma (LMP) 0.2815   4+  1444 s adenoma (LMP) 0.5005  4+  1447 s adenoma (LMP) 0.3741   4+  1450 s adenoma (LMP) 0.2195  4+  1036 m adenoma (LMP) 0.0775   0 1451 m adenoma (LMP) 0.0515   01456 m adenoma (LMP) 0.2771   4+  1242 s carcinoma 0.1502   2+  515 scarcinoma 1.5101   4+  1032 s carcinoma 0.28   4+  1240 s carcinoma1.2282   4+  1245 s carcinoma 0.9342   4+  465 s carcinoma 0.8166   4+ 1026 s carcinoma 0.4244   4+  464 s carcinoma 0.0827   0 468 s carcinoma0.5117   4+  1033 s carcinoma 0.036   0 1039 s carcinoma 0.1749   2+ 960 s carcinoma 0.3775   4+  962 s carcinoma 0.446   4+  1819 scarcinoma 0.5974   4+  1443 m carcinoma 0.1122   0 1219 m carcinoma0.8489   4+  1990 m carcinoma 1.3723   4+  484 m carcinoma 0.0007   01244 m carcinoma 0.0376   0 1816 m carcinoma 0.4697   4+  2295 ecarcinoma 0.2437   4+  2299 e carcinoma 0.4789   4+  2300 e carcinoma0.4094   4+  947 c carcinoma 0.5468   4+  948 c carcinoma 0.5086   4+ sw626 ovarian cancer 0.0633   0 cell line caov3 ovarian cancer 0.1862  2+  cell line Mean 0.05245   Confidence level (95.0%) 0.02769603Standard Error 0.01224319 Median 0.04165   Mode    #Num! StandardDeviation 0.03871635 Mean + 2SD    0.12989 = 2+ Mean + 4SD    0.20733 =4+ Sample Variance 0.00149896 Kurtosis −1.60048588   Skewness 0.37295213Range 0.1025   Minimum 0.0033   Maximum 0.1058   Sum 0.5245   Count10        

EXAMPLE 10

[0048] Statistical Analysis Semi-quantitative PCR Results

[0049]FIG. 3 summarizes the relative expression ratios ofantileukoprotease to β-tubulin in normal ovaries, LMP tumors, andovarian carcinomas. The exact ratios (mean ±SD) are listed in Table 2for normal ovary (0.05±0.04), LMP tumor (0.24±0.16) and carcinoma(0.50±0.41). In many carcinoma cases as well as LMP tumors,antileukoprotease mRNA expression was significantly elevated compared tothat in normal ovary (LMP tumor, p<0.01; carcinoma, p<0.01; unpairedT-test). There was no statistical difference between theantileukoprotease expression levels and the clinical stage, histologicalgrade or histological type. It should be noted that in one ovariancancer cell line, CaOv3, significant overexpression of antileukoproteasewas observed (ratio=0.19), while another cell line, SW626, showed arelatively low level of antileukoprotease expression (ratio=0.06) (Table1). TABLE 2 Relative Expression Levels of ALP and ALP OverexpressionRates in Ovarian Tumors Ratio ALP ALP/β-tubulin overexpression TissueType N Mean SD rates^(a) Normal Ovary 10 0.05 0.04 0/10 (0%)  LMP Tumor9  0.24^(b) 0.16 6/9 (67%) Serous 6 0.29 0.16  5/6 (83%)^(d) Mucinous 30.14 0.12 1/3 (33%) Ovarian Cancer 25  0.50^(c) 0.41 20/25 (80%) Clinical Stage Stage 1/2 7 0.54 0.45  6/7 (8.6%) Stage 3 18 0.49 0.4114/18 (78%)  Histological Grade Grade 1/2 13 0.62 0.52 10/13 (77%) Grade 3 12 0.38 0.22 10/12 (83%)  Histological Type Serous 14 0.54 0.4412/14 (86%)  Mucinous 6 0.47 0.55 3/6 (50%) Endometrioid 3 0.38 0.12 3/3 (100%) Clear Cell 2 0.53 0.03  2/2 (100%)

[0050] Table 2 also summarizes the mean antileukoprotease overexpressionrates by tissue subtypes in ovarian tumors. Overall, antileukoproteasemRNA overexpression was found in 6 of 9 LMP tumors (67%) and in 20 of 25carcinoma cases (80%). All 10 normal ovaries showed relatively lowlevels of antileukoprotease mRNA expression. With regard to histologicaltype, increased expression of antileukoprotease was found in 5 of 6 LMPserous tumors (83%) and in 12 of 14 serous carcinomas, whereas only 1 of2 LMP mucinous tumors (33%) and 3 of 6 mucinous carcinomas (50%) showedoverexpression of antileukoprotease. Thus, there was a statisticaldifference of the antileukoprotease overexpression rates between seroustumors (17/20) and mucinous tumors (4/9) including LMP tumors andcarcinomas (p<0.05, χ² test). The antileukoprotease overexpression ratesdetermined for clinical stage and histological grad did not show anysignificant difference.

[0051] Table 3 indicates the relationship between antileukoprotease andSCCE overexpression status in ovarian tumor cases including LMP tumorsand carcinomas. SCCE overexpression status was analyzed in the sameseries of the samples. Out of twenty-six antileukoproteaseoverexpression cases, 23 cases showed SCCE overexpression, whereas onlytwo out of eight cases showed SCCE overexpression with normal expressionof antileukoprotease. Thus, there was a significant positive correlationbetween antileukoprotease and SCCE overexpression status in ovariantumor cases (p<0.01, χ² test). TABLE 3 ALP and SCCE OverexpressionStatus in Ovarian Tumors Including LMP Tumors and Carcinomas SCCE mRNAExpression SCCE SCCE Overexpression^(a) Normal Range^(b) ALP mRNAExpression Cases Cases ALP Overexpression^(a) Cases 23 3 ALP NormalRange^(b) Cases 3 6

EXAMPLE 11

[0052] Immunohistochemical Analysis

[0053] To further confirm the presence of the antileukoprotease proteinin ovarian tumor cells. Antileukoprotease expression in both normalovarian epithelia and ovarian tumor tissues was analyzed byimmunohistochemical staining with the polyclonal serum describe supra. Acomparison of immunohistochemical staining of normal ovary and varioussub-types of ovarian carcinoma is shown in FIGS. 4A-4G. Differentpatterns of staining were noted in tumor cell cytoplasm, vesicles andmembranes. In a normal endocervical specimen, which was used as apositive control, antileukoprotease protein was detected in endocervicalglandular cells as well as on the mucin in the gland (FIG. 4A). Littleor no staining was observed with normal ovarian surface epithelium (FIG.4B). However, positive staining was observed both in the cytoplasm andon the cell membrane of ovarian cancer cells (FIG. 4C; (endometrioidadenocarcinoma).

[0054] Positive antileukoprotease staining was observed on the mucin aswell as in the tumor glands of mucinous carcinoma (FIG. 4D). Thesecretion of clear cell carcinomas also stained positive forantileukoprotease (FIG. 4E). The extracellular location ofantileukoprotease was especially detectable in endometrioid and clearcell tumors (FIGS. 4C and 4E respectively). Serous carcinomas were alsopositive for ALP immunohistochemical staining (FIGS. 4F and 4G). Morethan 10% positive tumor cell staining of ALP was detected in one of two(50%) benign adenomas, two (100%) of two LMP tumors, and 17 of 25 (68%)adenocarcinomas while little or no staining was observed in all threenormal ovary specimens. A more complete analysis of theimmunohistochemical findings is presented in Table 4. 9 out of 12borderline and low malignant potential tumors stained positively forantileukoprotease while 12 out of 19 carcinomas showed positivestaining. TABLE 4 A complete analysis of ALP immunohistochemicalfindings Case Stage Histology Grade ALP Prognosis 1 normal ovary 0− 2normal ovary 0− 3 normal ovary 0− 4 mucinous B 0− Alive 5 mucinous B 2+Alive 6 1a serous LMP G1 1+ Alive 7 1a mucinous LMP G1 1+ Alive 8 1amucinous ca G1 1+ weak Alive 9 1a mucinous ca G2 0− Alive 10 1aendometrioid ca G1 2+ Alive 11 1c serous ca G1 1+ Alive 12 1c mucinousca G1 2+ Alive 13 1c mucinous ca G1 2+ Alive 14 1c clear cell ca G2 1+Alive 15 1c clear cell ca G2 0− Alive 16 2c serous ca G3 1+ Alive 17 3amucinous ca G2 2+ Alive 18 3b serous ca G1 2+ Alive 19 3c serous ca G10− Dead 20 3c serous ca G3 0− Alive 21 3c serous ca G2 2+ Alive 22 3cserous ca G1 2+ unknown 23 3c serous ca G3 0− Alive 24 3c serous ca G20− Dead 25 3c mucinous ca G1 2+ Dead 26 3c mucinous ca G2 2+ unknown 273c endometrioid ca G2 1+ Alive 28 3c endometrioid ca G1 0− Dead 29 3cendometrioid ca G2 0− Alive 30 3c endometrioid ca G2 1+ Dead 31 3cendometrioid ca G3 2+ Alive 32 3c clear cell ca G3 2+ Dead

EXAMPLE 12

[0055] Analysis and Implications of ALP Expression in Ovarian Tumors

[0056] In recent years, aberrant expression of serine proteases such asplasminogen activator has been shown to correlate positively with theinvasiveness and metastatic potential of tumor cells (9). Moresignificantly, the serine protease known as prostate-specific antigen(PSA) has been used successfully as a tumor marker for the earlydiagnosis of prostate cancer (23). Serine proteases play important rolesin the cascade of events involved in the malignant processes, and atleast for prostate cancer, provide sufficient signal to allow detectionof early disease.

[0057] Specific inhibitors for most of the proteolytic enzymes have beenidentified and it has been contemplated that these inhibitors inhibitextracellular degradation, which in turn prevents tumor cell invasion.For example, plasminogen-activator-inhibitor 1 is suggested to protectthe tumor stroma from ongoing urokinase-plasminogen-activator mediatedproteolysis in many human tumors (24). The proteolytic activityassociated with tumors is probably a highly regulated cascade and theinterplay between proteases and their inhibitors may play a specificrole in tumor development and progression.

[0058] In the process of studying protease enzymes in ovarian tumors,several candidate genes have been identified to be overexpressed. In aneffort to identify other genes which are overexpressed early in thecarcinogenic progression of ovarian cancer, a screening strategy wasdeveloped using redundant primers to evolutionary conserved domains ofextracellular proteases such as the conserved catalytic triad domain ofthe serine protease family (viz. His--Asp--Ser). In the present study,antileukoprotease, a specific inhibitor of SCCE, was found in beexpressed in abundance in carcinoma tissues, with little or noexpression in normal ovary.

[0059] Antileukoprotease (ALP) is a mucosal secretory protein that hasbeen identified as a potent protease inhibitor of leukocyte serineproteases (18). Antileukoprotease is a secreted protein with adistribution of cysteines known to provide extraordinary stability toinhibitors. Immunocytochemical localization studies have revealed itspresence in respiratory tissues, salivary gland, cervical gland, andlacrimal gland (25). Moreover, antileukoprotease can be extracted fromthe human stratum corneum and is constitutively produced and releasedfrom human keratinocyte cell cultures (26). Therefore, Wiedow et al.(27) have suggested that antileukoprotease might not only regulateserine protease activities in mucus secretions but in skin as well.

[0060] Recently, Franzke et al. (17) reported that antileukoprotease isthe major inhibitor of SCCE in the epidermis and that it seems to beinvolved in the regulation of desquamation under physiological andpathological conditions. In vivo, SCCE mRNA is expressed in the upperspinous and granular layers of the epidermis (15, 16), and active SCCEcan be isolated from human horny layers (13). The fact that inhibitionof SCCE causes the concomitant complete inhibition of cell shedding fromplantar stratum corneum (desquamation) in vitro (28) led to thehypothesis that SCCE may be involved in the process of physiologicaldesquamation (13-16,28). The fact that SCCE is overexpressed in ovariantumors supports the potential of SCCE as a target for inhibition of downregulation in therapeutic interventions aimed at preventing the spreador metastasis of ovarian cancer.

[0061] Herein, Northern blot hybridization has shown that theantileukoprotease transcript is abundant in ovarian carcinomas but isnot detected in normal ovaries. Semi-quantitative PCR analysis supportsthe observation that antileukoprotease mRNA levels are significantlyhigher in ovarian tumors as compared to normal ovaries. These resultswere confirmed by immunohistochemistry experiments which confirmed thatantileukoprotease is present in ovarian tumor cells and the mucinsecreted therefrom, whereas little or no staining is observed in normalovarian surface epithelium.

[0062] Positive correlation has been demonstrated between SCCE andantileukoprotease mRNA overexpression in ovarian tumor samples includingLMP tumors and carcinomas. This observation is, in one sense,paradoxical, since antileukoprotease levels would be expected to be lowif SCCE plays an important role during ovarian cancer development andprogression. However, the present results demonstrate thatco-transcriptional activation SCCE and antileukoprotease seems to occurduring transformation and initial tumor growth of ovarian cancer. Thesedata are entirely consistent with similar data observed for SCCE/ALPexpression in differentiated keratinocytes. In light of the fact thatdesquamation of skin cells is SCCE dependent and can be inhibited byALP, it is suggested that some dis-synchrony in time or space allowsSCCE activation in the presence of ALP.

[0063] The present findings show that the presence of SCCE andantileukoprotease together in tumor cells may similarly allow theshedding or desquamation of malignant cells through a similardis-synchrony. The fact that inhibition of SCCE activity prevents normaldesquamation of skin cells points to the potential of SCCE as a targetfor inhibition or down regulation of the spread or metastasis of ovariancarcinoma. Because antileukoprotease is a specific inhibitor of SCCE, itmay also be useful in the abatement of tumor growth, and progression inlow-antileukoprotease-expressing ovarian cancers, although there isalready high-level-ALP expression observed in many ovarian cancers.

[0064] It has been demonstrated herein that the overexpression ofantileukoprotease is a common event in ovarian tumors. Becauseantileukoprotease is a secreted protein and antileukoprotease appears inabundance only in tumor tissues as demonstrated by Northern blotanalysis and semi-quantitative analyses, it has a potential for beingpresent in the circulation of tumor-bearing patients. The overexpressionof antileukoprotease in LMP tumors and stage I carcinomas is ofparticular note as the antileukoprotease is produced directly by tumorcells instead of underlying stromal tissues. As a result of this, assaysmay be developed for the early detection of ovarian cancer based on thedetection the antileukoprotease protein. This molecule fulfills manycriteria necessary for a valuable marker of tumor growth andprogression. It is overexpressed in tumor cells secreted to theextracellular space and is a relatively small protein (approximately 100amino acids) providing good opportunity for uptake into the circulation.Moreover, antileukoprotease is a very stable protein providing potentialfor a relatively long half life in the circulation. In addition, eventhough antileukoprotease has been shown to directly inhibit SCCEactivity, it co-existence with SCCE during desquamation of keratinocytessuggests that a similar mechanism of antileukoprotease sequestration oflack of ability to inhibit SCCE may allow the desquamation or sheddingof ovarian tumor cells.

[0065] The following references were cited herein.

[0066] 1. Neurath, H. The diversity of proteolytic enzymes. In: Beynonet al., (eds.), Proteolytic enzymes, Oxford, IRL Press 1989:1-13.

[0067] 2. Liotta, et al., Cell 1991; 64:327-336.

[0068] 3. Duffy, M. J. Clin Exp Metastasis 1992; 10:145-155.

[0069] 4. Tryggvason, et al., Biochem Biophys Acta 1987; 907:191-217.

[0070] 5. Powell, W. C. et al., Cancer Res 1993, 53:417-422.

[0071] 6. MacDougall, J. R. et al., Cancer and Metastasis Reviews 1995;14:351-362.

[0072] 7. Rempel, S. A. et al., Cancer Res 1994; 54:6027-6031.

[0073] 8. Nazeer, T. et al., Am J Clin Pathol 1992; 97:764-769.

[0074] 9. Duffy, M. J. et al., Cancer (Phila) 1988; 62:531-533.

[0075] 10. Liotta, L. A. et al., Cell 1991; 64:327-336.

[0076] 11. Wang, M. et al., Cancer Res 1988; 48:6262-6271.

[0077] 12. Reich, R. et al., Cancer Res 1988; 48:3307-3312.

[0078] 13. Egelrud, T. J Invest Dermatol 1993; 101:200-204.

[0079] 14. Hansson, et al., J Biol Chem 1994;269:19420-19426.

[0080] 15. Sondell, et al., J Histochem Cytochem 1994;42:459-465.

[0081] 16. Sondell, et al., J Invest Dermatol 1995; 104:819-823.

[0082] 17. Franzke, et al., J Biol Chem 1996;271:21886-21890.

[0083] 18. Thompson, R. C., et al., Proc Natl Acad Sci USA 1986;83:6692-6696, 1986.

[0084] 19. Shigemasa, et al., J Soc Gynecol Invest 1997;4:95-102.

[0085] 20. Tanimoto, et al., Gynecol Oncol 1997;66:308-312.

[0086] 21. Shigemasa, K. et al., Int J. Gynecol Cancer 1997,7(4):296-303.

[0087] 22. Hall, et al., Mol Cell Biol 1983; 3:854-862.

[0088] 23. McCormack, R. T. et al., Urology 1995; 45:729-744.

[0089] 24. Pappot et al., Biol Chem Hoppe Seyler 1995; 376(5):259-267

[0090] 25. Franken C. et al., J Histochem Cytochem 1989; 37:493-498.

[0091] 26. Wiedow, O. (1995) Isolierung und Charakterisierung vonSerinprotease Inhibitoren der menschlichen Epidermis, Köster, Berlin

[0092] 27. Wiedow, O. et al., J Invest Dermatol 1993; 101:305-309.

[0093] 28. Lundstrom, A. et al., J Invest Dermatol 1988; 91:340-343.

[0094] 29. Garver, R. I. et al., Gene Ther 1994; 1:46-50.

[0095] 30. Lunderström, et al.,. (1988) J. Invest. Dermatol. 101,200-204.

[0096] Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically incorporated by reference.

[0097] One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

1 6 1 17 DNA Artificial sequence Forward oligonucleotide primer foramplification of antileukoprotease 1 tgcattgaca acgaggc 17 2 17 DNAArtificial sequence Reverse oligonucleotide primer for PCR amplificationof antileukoprotease 2 ctgtcttgac attgttg 17 3 20 DNA Artificialsequence Forward oligonucleotide primer for PCR amplification ofantileukoprotease 3 ccttcaaagc tggagtctgt 20 4 20 DNA Artificialsequence Reverse oligonucleotide primer for PCR amplification ofantileukoprotease 4 ccaaaggagg atatcagtgg 20 5 20 DNA Artificialsequence Forward oligonucleotide primer for PCR amplification of(-tubulin 5 cgcatcaacg tgtactacaa 20 6 20 DNA Artificial sequenceForward oligonucleotide primer for PCR amplification of (-tubulin 6tacgagctgg tggactgaga 20

What is claimed is:
 1. A method of detecting growth of an ovarian tumorand ovarian-derived metastatic tumor in a sample from an individual,comprising the step of: measuring the level of antileukoprotease in saidindividual, wherein if said level of antileukoprotease exceeds the meanbasal level of antileukoprotease in nondiseased individuals by 2standard deviation or greater, said individual has growth in an ovarianor ovarian-derived tumor.
 2. The method of claim 1, wherein said tumoris selected from the group consisting of a low malignant potential tumorand an ovarian carcinoma.
 3. The method of claim 2, wherein said ovariancarcinoma is selected from the group consisting of a serous carcinoma, amucinous carcinoma, an endometrioid carcinoma and a clear cellcarcinoma.
 4. The method of claim 1, wherein said antileukoprotease isdetected in a sample selected from the group consisting of an ovariansecretion, an ovarian biopsy, a metastatic tumor biopsy and blood. 5.The method of claim 1, wherein said antileukoprotease is detected by amethod selected from the group consisting of western blot analysis,immunohistochemical staining, and immunochemical methods.
 6. A method oftreating an individual having a tumor selected from the group consistingof an ovarian tumor and ovarian-derived metastatic tumors, comprisingthe step of: administering antileukoprotease to said individual.
 7. Themethod of claim 5, wherein said tumor is selected from the groupconsisting of a low malignant potential tumor and an ovarian carcinoma.8. The method of claim 7, wherein said ovarian carcinoma is selectedfrom the group consisting of a serous carcinoma, a mucinous carcinoma,an endometrioid carcinoma and a clear cell carcinomas.
 9. The method ofclaim 6, wherein said antileukoprotease is administered systemically.10. The method of claim 6, wherein said antileukoprotease isadministered locally
 11. A method of preventing metastasis of a tumorselected from the group consisting of an ovarian tumor and ovarianderived metastatic tumors, comprising the step of: administeringantileukoprotease to an individual having said tumor.
 12. The method ofclaim 11, wherein said tumor is selected from the group consisting of alow malignant potential tumor and an ovarian carcinoma.
 13. The methodof claim 12, wherein said ovarian carcinoma is selected from the groupconsisting of a serous carcinoma, a mucinous carcinoma, an endometrioidcarcinoma and a clear cell carcinoma.
 14. The method of claim 11,wherein said antileukoprotease is administered systemically.
 15. Themethod of claim 11, wherein said antileukoprotease is administeredlocally